Copper is an essential trace element with a critical role in the biochemistry of cellular respiration, iron homeostasis and antioxidant defense. The long-term objective of these studies is to define the cellular and molecular determinants of human copper metabolism. Recent studies have revealed that the delivery of copper to specific pathways within the cell is mediated by a unique class of intracellular carrier proteins termed copper chaperones. The studies in this proposal are intended to elucidate the role of the copper chaperone for superoxide dismutase (CCS) in intracellular copper homeostasis and in the pathogenesis of familial amyotrophic lateral sclerosis (FALS) in patients with inherited mutations in Cu/Zn superoxide dismutase (SOD1). Structure/function studies will be accomplished using site-directed mutagenesis and expression of CCS in cells from mice with a germline deletion of the CCS gene. The interaction of CCS and SOD1 in intact, living single cells will be analyzed using blue and green fluorescent fusion proteins and fluorescence resonance energy transfer microscopy. The molecular and cellular mechanisms determining copper trafficking to CCS from cytoplasmic or storage sites will be elucidated by identification and characterization of novel CCS interacting proteins. Finally, the precise role of CCS in the pathogenesis of FALS will be evaluated by examining disease onset and progression in FALS SOD1 transgenic mice bred onto the genetic background of CCS deficiency. Taken together the results of these studies will permit new insights into the mechanisms of intracellular copper homeostasis and may allow for novel nutritional strategies to prevent or ameliorate human disease. The long-range objective of these studies is to define the cellular and molecular determinants of human copper metabolism. Four specific aims are identified: 1. To perform a detailed structural and functional analysis of the copper chaperone for superoxide dismutase (CCS). 2. To define the spatial and temporal interaction of CCS and SOD1 in living cells. 3. To elucidate the mechanisms of intracellular copper trafficking to CCS. 4. To examine the role of CCS in the pathogenesis of neuronal degeneration in familial amyotrophic lateral sclerosis (FALS).